Dipeptidyl peptidase IV (DPIV) is a serine protease which cleaves N-terminal dipeptides from a peptide chain containing, preferably, a proline residue in the penultimate position. Although the biological role of DPIV in mammalian systems has not been completely established, it is believed to play an important role in neuropeptide metabolism, T-cell activation, attachment of cancer cells to the endothelium and the entry of HIV into lymphoid cells.
Likewise, it has been discovered that DPIV is responsible for inactivating glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide also known as gastric-inhibitory peptide (GIP). Since GLP-1 is a major stimulator of pancreatic insulin secretion and has direct beneficial effects on glucose disposal, in WO 97/40832 and U.S. Pat. No. 6,303,661 inhibition of DPIV and DPIV-like enzyme activity was shown to represent an attractive approach for treating non-insulin-dependent diabetes mellitus (NIDDM).
The present invention provides a new use of DPIV-inhibitors for the treatment of conditions mediated by inhibition of DPIV and DPIV-like enzymes, in particular the treatment of cancer and tumors and the inhibition of metastasis and tumor colonization, and pharmaceutical compositions e.g. useful in inhibiting DPIV and DPIV-like enzymes and a method of inhibiting said enzyme activity.
This invention relates to a method of treatment, in particular to a method for the treatment of cancer, tumors, metastasis and tumor colonization and to compositions for use in such method. Dipeptidyl peptidase IV (DPIV) is a post-proline (to a lesser extent post-alanine, post-serine or post-glycine) cleaving serine protease found in various tissues of the body including kidney, liver, and intestine.
It is known that DPIV-Inhibitors may be useful for the treatment of impaired glucose tolerance and diabetes mellitus (International Patent Application, Publication Number WO 99/61431, Pederson R A et al, Diabetes. 1998 August; 47(8):1253–8 and Pauly R P et al, Metabolism 1999 March; 48(3):385–9). In particular WO 99/61431 discloses DPIV-Inhibitors comprising an amino acid residue and a thiazolidine or pyrrolidine group, and salts thereof, especially L-threo-isoleucyl thiazolidine, L-allo-isoleucyl thiazolidine, L-threo-isoleucyl pyrrolidine, L-allo-isoleucyl thiazolidine, L-allo-isoleucyl pyrrolidine, and salts thereof.
Further examples of low molecular weight dipeptidyl peptidase IV inhibitors are agents such as tetrahydroisoquinolin-3-carboxamide derivatives, N-substituted 2-cyanopyroles and -pyrrolidines, N-(N′-substituted glycyl)-2-cyanopyrrolidines, N-(substituted glycyl)-thiazolidines, N-(substituted glycyl)-4-cyanothiazolidines, amino-acyl-borono-prolyl-inhibitors, cyclopropyl-fused pyrrolidines and heterocyclic compounds. Inhibitors of dipeptidyl peptidase IV are described in U.S. Pat. No. 6,380,398, U.S. Pat. No. 6,011,155; U.S. Pat. No. 6,107,317; U.S. Pat. No. 6,110,949; U.S. Pat. No. 6,124,305; U.S. Pat. No. 6,172,081; WO 95/15309, WO 99/61431, WO 99/67278, WO 99/67279, DE 198 34 591, WO 97/40832, DE 196 16 486 C 2, WO 98/19998, WO 00/07617, WO 99/38501, WO 99/46272, WO 99/38501, WO 01/68603, WO 01/40180, WO 01/81337, WO 01/81304, WO 01/55105, WO 02/02560 and WO 02/14271, the teachings of which are herein incorporated by reference in their entirety.
The term DPIV-like enzymes relates to structurally and/or functionally DPIV/CD26-related enzyme proteins (Sedo & Malik, Dipeptidyl peptidase IV-like molecules: homologous proteins or homologous activities? Biochimica et Biophysica Acta 2001, 36506: 1–10). In essence, this small group of enzymes has evolved during evolution to release H-Xaa-Pro-Dipeptides and H-Xaa-Ala-Dipeptides from N-terminus of oligo- or polypeptides. They show the common feature, that they accomotate in the Pro-position also Al, Ser, Thr and other amino acids with small hydrophobic side-chains as, Gly or Val. The hydrolytic efficacy is ranked Pro>Ala>>Ser, Thr>>Gly, Val. Same proteins have been only available in such small quantities, that only the post-Pro or post-Ala cleavage could be established. While the proteins: DPIV, DP II, FAPα (Seprase), DP 6, DP 8 and DP 9 are structurally related and show a high sequence homology, attractin is an extraordinary functional DPIV-like enzyme, characterized by a similar activity and inhibitory pattern.
Further DPIV-like enzymes are disclosed in WO 01/19866, WO 02/04610, WO 02/34900 and WO 02/31134. WO 01/19866 discloses novel human dipeptidyl aminopeptidase (DPP8) with structural und functional similarities to DPIV and fibroblast activation protein (FAP). WO 02/04610 provides reagents, which regulate human dipeptidyl peptidase IV-like enzyme and reagents which bind to human dipeptidyl peptidase IV-like enzyme gene product. These reagents can play a role in preventing, ameliorating, or correcting dysfunctions or diseases including, but not limited to, tumors and peripheral and central nervous system disorders including pain and neurodegenerative disorders. The dipeptidyl peptidase IV-like enzyme of WO 02/04610 is well known in the art. In the Gene Bank data base, this enzyme is registered as KIAA1492 (registration in February 2001, submitted on Apr. 4, 2000, AB040925). In the Merops data base, the dipeptidyl peptidase IV-like enzyme of WO 02/04610 is registered as non-protease homologue. The Merops homologue of the dipeptidyl peptidase IV-like enzyme disclosed in WO 02/04610 and the active site motive thereof was confirmed by the human genome project. WO 02/34900 discloses a novel dipeptidyl peptidase 9 (DPP9) with significant homology with the amino acid sequences of DPIV and DPP8. WO 02/31134 discloses three DPIV0-like enzymes. DPRP1, DPRP2 and DPRP3. Sequence analysis revealed, that DPRP1 is identical to DPP8, as disclosed in WO 01/19868, that DPRP2 is identical to DPP9 and that DPRP3 is identical to KIAA1492 as disclosed in WO 02/04610.
DPIV and DPIV-Like Enzymes in Immunophysiology and Cancer
Dipeptidyl peptidase IV (DPIV; EC 3.4.14.5; CD26) CD26 is a M r 110,000 surface glycoprotein with an array of diverse functional properties that is expressed on a number of tissues, including epithelial cells and leukocyte subsets (Mentlein, 1999). Furthermore, it is a membrane-associated ectopeptidase that possesses DPIV-like activity in its extracellular domain and is able to cleave N-terminal dipeptides from polypeptides with either L-proline or L-alanine in the penultimate position. In general, DPIV is recognized as an ectopeptidase with a triple functional role. DPIV is involved in catalyzing the release of Xaa-Pro dipeptides from circulating hormones and chemokines (De Meester et al, 1999; Mentlein, 1999), in T cell dependent immune responses (Kähne et al, 1999; Korom et al, 1997), and in cell adhesion including metastasis (Mentlein, 1999).
In addition DPIV has been identified as the ADA binding protein, thereby regulating ADA surface expression, with the DPIV/ADA complex perhaps playing a key role in the catalytic removal of local adenosine to regulate immune system function. Besides being a key immunoregulatory molecule, DPIV may have a potential role in the development of certain neoplasms (Mattern et al., 1993; Carbone et al., 1995). In eukaryotic cells, cell cycle progression is controlled at the G1-S checkpoint by a group of related enzymes known as the CDKs, which are positively regulated by their physical association with regulatory subunits called cyclins. It has been demonstrated that binding of soluble anti-CD26 antibodies inhibits the growth of anaplastic large cell T-cell lymphoma cell lines, both in in vitro and in vivo experiments (Ho et al., 2001).
Cancer Pathomechanisms
Cancer is a group of over 150 diseases characterized by the uncontrolled growth of abnormal cells in the body. Normal cells can become abnormal when they are exposed to carcinogens such as radiation or particular drugs or chemicals. They can also turn malignant (cancerous) when they are attacked by certain viruses or when some not-yet-fully-understood internal signal occurs. Once cells become malignant, they multiply more rapidly than usual. Then they often form masses called tumors that invade nearby tissue and interfere with normal bodily functions. Cancer cells also have a tendency to spread to other parts of the body, where they may form a secondary tumor.
Mechanisms of Metastasis
The outcome of cancer metastasis depends on multiple interactions within the target tissue and depends on the microenvironment including cellular adhesion molecules (Carlos, 2001), chemokines (Muller et al., 2001), or hydrodynamic effects (Haier and Nicholson, 2001) and many other factors (Fidler, 2001). In addition, a very rapid attraction of leukocytes and specific cellular responses at the tumor sites may play a critical role in the early host defense against cancer (Shingu et al.; 2002). These early changes may be of critical importance for the outcome of metastatic disease and may extend the present understanding of the host resistance against metastasis.
DPIV and DPIV-Like Enzymes and Tumor Adhesion and Colonization
For cancer cell or metastatic cell adhesion, a prominent expression of DPIV on endothelia of lung capillaries accounts for arrest of blood borne breast cancer cells (Johnson et al, 1993). Fibronectin (FN) and probably also collagen collected on the breast cancer cell surface were identified as the principal ligands for DPIV (Abdel-Ghany et al, 1998; Cheng et al, 1998).
Ho and colleagues (2001) show that binding of soluble anti-CD26 monoclonal Ab 1F7 inhibits the growth of the human CD301 anaplastic large cell T-cell lymphoma cell line Karpas 299 in both in in vitro and in vivo experiments. In vitro experiments show that 1F7 induces cell cycle arrest at the G1-S checkpoint, associated with enhanced p21 expression that is dependent on de novo protein synthesis. Furthermore, experiments with a severe combined immunodeficient mouse tumor model demonstrate that 1F7 treatment significantly enhances survival of tumor-bearing mice by inhibiting tumor formation.
Protease Inhibitors, Antibodies and Proteases as Anti-Tumor Agents
WO 95/29691 discloses proline phosphonate derivatives as inhibitors of serine proteases with chymotrypsin-like, trypsin-like, elastase-like and dipeptidyl peptidase IV specificity and their roles as anti-inflammatory agents, anticoagulants, anti-tumor agents and anti-AIDS agents.
WO 98/53812 and WO 97/48409 disclose novel methods of using phosphonate derivatives, hydroxyphosphinyl derivatives, and phosphoramidate derivatives to inhibit N-Acetlyated α-Linked Acidic Dipeptidase (NAALADase) enzyme activity, and to treat prostate diseases, especially using these compounds for the inhibition of prostate cancer cell growth.
WO 01/92273 discloses new benzenedicarboxylic acid derivative compounds, pharmaceutical compositions, diagnostic methods and diagnostic kits that include those compounds and methods of using those compounds for inhibiting NAALADase enzyme activity, detecting diseases where NAALADase levels are altered, affecting neuronal activity, affecting TGF-β activity, inhibiting angiogenesis and treating glutamate abnormalities, neuropathy, pain, compulsive disorders, prostate diseases, cancer and glaucoma.
WO 01/34596 discloses pyrrolecarbonylimino derivatives, pharmaceutical compounds and methods of using those compounds to inhibit NAALADase enzyme activity, thereby affecting neuronal activities, inhibiting angiogenesis and treating glutamate abnormalities, compulsive disorders, prostate diseases and cancer.
WO 00/71135 discloses a method for treating subjects with abnormal cell proliferation. The method involves administering to subjects in need of such treatment an effective amount of boro-proline compounds, to inhibit cell proliferation such as that associated with tumor growth and metastasis. A method for inhibiting angiogenesis in an abnormal proliferative cell mass by the administration of a boro-proline derivative is also provided. The invention of WO 00/71135 is based, in part, on the observation, that the boro-proline derivatives are able to inhibit the enzymatic activity of fibroblast activation protein-alpha (FAP-α).
WO 00/71571 relates to a prodrug that is capable of being converted into a drug by the catalytic action of human fibroblast activation protein-alpha (FAP-α), said prodrug having a cleavage site which is recognised by FAP-α, and said drug being cytotoxic or cytostatic under physiological conditions. These prodrugs are converted into a drug at the site of the tumor.
WO 00/10549 discloses compounds and a method for regulation of substrate activity in vivo useful for the treatment of medical disorders such as arteriosclerosis, allergies, inflammation, angiogenesis, cardiogenesis, neoplasm, tumor, cancer, a hepatic disease, an intestinal disease, organ vascularization, and microbial and viral infections. The compounds consist of a targeting moiety that binds to DPIV, and a reactive group, that reacts at a reactive center of DPIV. Said compounds are used to prevent chemokine alteration by inhibiting DPIV activity.
WO 00/36420 discloses a method for identifying nucleotide sequences that are differentially expressed in tumor cells, preferably primary breast tumor cells, comprising exposing a tumor cell containing tissue sample to an agent specific for fibroblast activation protein (FAP) separating cells recognised by said agent from the remaining cells in the sample and harvesting said remaining cells. Nucleic acid molecules derived from the use of this technique are also described, together with compositions comprising the same and their uses in pharmaceutical compositions for treating a disease, preferably breast cancer.
WO 99/47152 discloses a method of suppressing the malignant phenotype or inducing apoptosis of cancer cells in a subject, comprising introducing into the cancer cell an amount of a nucleic acid encoding a dipeptidyl peptidase IV protein or fibroblast activating protein-α, thereby suppressing the malignant phenotype of the cancer. WO 99/47152 also discloses a method of inducing expression of dipeptidyl peptidase IV or fibroblast activating protein-α in cancer cells of a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of an agent capable of activating transcription of the dipeptidyl peptidase IV gene or fibroblast activating protein-α gene and a pharmaceutical acceptable carrier or diluent.
WO 01/74299 discloses antibodies that specifically bind to a membrane protease complex, the complex consisting of two homodimers of seprase and dipeptidyl peptidase IV (DPIV), obtained from mammalian, preferably human cell membranes. The antibodies specifically bind to the DPIV protease of the seprase-DPIV complex. This membrane protease complex resides on cell surface invadopodia at the leading edge of angiogenic endothelia, migratory fibroblasts, and invading cancer cells.
WO 02/20825 relates to novel methods and compositions for detection and isolation of cancer cells with metastatic potential. WO 02/20825 further relates to assays for measuring the metastatic potential of such cancer cells and drug screening assays for the indentification of agents having anti-metastatic potential. Also disclosed are methods and compositions for inhibiting the metastatic potential of cancer cells by modulating the activity of serine integral membrane proteases [(SIMP) consisting of seprase and dipeptidyl peptidase IV (DPIV)] expressed on the surface of metastasizing cancer cells, by using antibodies against SIMP.
Current Treatments of Cancer and Tumor Cell Adhesion
Current cancer treatment regimens comprise surgery, chemotherapy, radiation therapy, and other treatment methods including immunotherapy. Immunotherapy is composed of the usage or the modification of intrinsic bodily mechanisms—in most cases immune mechanisms—to fight cancer. Chemotherapy kills cancer cells through the use of drugs or hormones. Taken either orally or through injection, chemotherapeutic agents are used to treat a wide variety of cancer. They may be given alone or in combination with surgery or radiation or both. Chemotherapy is an established way to destroy hard-to-detect cancer cells that have spread and are circulating in the body. Anemia (low number of red blood cells) is a frequent side effect of chemotherapy and may cause symptoms such as extreme tiredness, dizziness, or shortness of breath. Epoetin alfa (Procrit®, Epogen®)—recombinant erythropoietin that stimulates red blood cell production—is a prescription drug available for the treatment of chemotherapy-related anemia.
Immunotherapy uses the body's own immune system or other parts of the organism to destroy cancer cells. This form of treatment is still being intensively studied in clinical trials; it is not yet widely available to most cancer patients. The various immunological agents used include substances produced by the body (such as the interferons, the interleukins and tumor necrosis factor) and laboratory-produced substances (such as monoclonal antibodies and vaccines). Immunological agents work in different ways and can be used independently or in combination with other forms of treatment.
Angiogenesis Inhibitors as Anti-Metastatic Drugs in Immunotherapy
Angiogenesis inhibitors are drugs that block the development of new blood vessels. Solid tumors cannot grow without inducing the formation of new blood vessels. Blocking the development of new blood vessels cuts off the tumor's supply of oxygen and nutrients.
Several angiogenesis inhibitors are currently being tested in human trials. In cancerous tissue, tumors cannot grow or spread (metastasize) without the development of new blood vessels. Blood vessels supply tissues with oxygen and nutrients necessary for survival and growth.